The long-range goal of this project is to define the interactionsin the transcriptioncomplex that regulate pausing and termination by RNA polymerase. Nascent RNA hairpins are important regulatory signals in bacteria, where pausing and termination are major components of genetic regulatory mechanisms. Pausing and premature termination also affect expression of genes in mammalian cells and viruses, notably genes involved in the development of cancer and in growth of the AIDS virus, HIV-1. In both bacteria and eukaryotes, specialized regulatory proteins modify the transcriptioncomplex to make it resistant to pausing and termination. Although significant progress has been made in understandingpausing, termination, and the regulatory proteins that control these events, two alternativemodels remain possible. In one view, called the allosteric model, pause signals, termination signals, and regulatory proteins primarily affect the [unreadable]conformation of RNA polymerase. In the other, these signals and proteins primarily affect translocation of a relatively rigid RNA polymerase on the RNA and DNA chains (the rigid-body model). Pausing and termination by E. coli RNA polymerase and their regulation by the NusA, NusG, and RfaH proteins, and pausing by human RNA polymerase II have been developed as model systems. A combination of biochemical, genetic, and biophysical approaches will be used to distinguish the allosteric and rigid-body models of transcriptional regulation, and to characterize the mechanisms of pausing, termination, and regulatory proteins that control them. Specific aims will be to (i) characterize interactions of RNA polymerase's flap-tip helix with RNA, NusA, and a70, and test how these interactions affect catalysis in the active site;(ii) determine the location of the RNA 3'end in paused and nonpaused transcription elongation complexes;(Hi)determine the kinetic mechanisms of elongation, pausing, and termination;(iv) map interactions between RNA polymerase and pause and terminator hairpins;and (v) determine the sites at which RfaH and NusG interact with RNA polymerase and the mechanisms by which they regulate transcript elongation. E si Ib(S) (organization, city, state) University of Wisconsin, Madison, WI Rockefeller University, New York, NY Brandeis University, Waltham, MA Stanford University, Stanford, CA St. Louis University, St. Louis, MO KEY PERSONNEL. See instructions on Page 11. Name Robert Landick Irina Artsimovitch Innokenti Toulokhonov Murali Palangat Kati Geszvain Christina Farmer Rachel Mooney Seth Darst JeffGelles Steve Block Tomasz Heyduk Use continuation pages as needed to provide the required information in the format shown below. Organization Role on Project University of Wisconsin-Madison Principal Investigator University of Wisconsin-Madison Postdoctoral Researcher University of Wisconsin-Madison Postdoctoral Researcher University of Wisconsin-Madison Postdoctoral Researcher University of Wisconsin-Madison Graduate Assistant University of Wisconsin-Madison Graduate Assistant University of Wisconsin-Madison Graduate Assistant Rockefeller University Collaborator Brandeis University Collaborator Stanford University Collaborator St. Louis University Collaborator PHS 398 (Rev. 4/98) Page 2 BB CC Principal Invesjjaator/Program Director (Last, first, middle): Roherf T .andirlr Type the name of the principal investigator/prograi^Hbctor at the top of each printed page and each con^Btion page. (For type specifications, see instructions on page 6.) RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page 1 Description,